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WO1999067391A9 - Origine de replication et ses methodes d'utilisation - Google Patents

Origine de replication et ses methodes d'utilisation

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Publication number
WO1999067391A9
WO1999067391A9 PCT/US1999/014487 US9914487W WO9967391A9 WO 1999067391 A9 WO1999067391 A9 WO 1999067391A9 US 9914487 W US9914487 W US 9914487W WO 9967391 A9 WO9967391 A9 WO 9967391A9
Authority
WO
WIPO (PCT)
Prior art keywords
oric
polynucleotide
sequence
polypeptide
seq
Prior art date
Application number
PCT/US1999/014487
Other languages
English (en)
Other versions
WO1999067391A1 (fr
Inventor
Earl W May
Original Assignee
Smithkline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corp filed Critical Smithkline Beecham Corp
Priority to JP2000556033A priority Critical patent/JP2002518049A/ja
Priority to EP99933582A priority patent/EP1088074A1/fr
Publication of WO1999067391A1 publication Critical patent/WO1999067391A1/fr
Publication of WO1999067391A9 publication Critical patent/WO1999067391A9/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides and oriC polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
  • the invention relates to polynucleotides and oriC polypeptides, hereinafter referred to as "oriC”.
  • Staphylococcal polynucleotides and gene products are particularly preferred by the invention to employ Staphylococcal polynucleotides and gene products as targets for the development of antibiotics.
  • the Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues. Staphylococcus aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci.
  • Staphylococcal food poisoning scalded skin syndrome
  • toxic shock syndrome The frequency of Staphylococcus aureus infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This phenomenon has created a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism.
  • polynucleotides of the invention possess amino acid sequence homology to known prokaryotic origins of replication. SUMMARY OF THE INVENTION
  • oriC polypeptides that have been identified as oriC polypeptides by binding to or modulating the activity (increasing or decreasing) of an oriC polynucleotide of the invention; these oriC polypeptides are herein referred to as an "oriC polypeptides(s)” or "oriC protein(s)” or “polypeptide(s)” or "binding molecules"
  • the polynucleotide comprises a region of oriC comprising a sequence set out in Table 1 [SEQ ID NO:l] which includes a full length origin of replication, or a variant thereof.
  • an oriC protein from Staphylococcus aureus, or a variant thereof.
  • oriC nucleic acid molecules particularly Staphylococcus aureus oriC nucleic acid molecules, including mRNAs, cDNAs, and genomic DNAs.
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
  • particularly preferred embodiments of the invention are naturally occurring allelic variants of oriC polynucleotides and oriC polypeptides.
  • oriC polypeptides of the invention there are provided oriC polypeptides of
  • Staphylococcus aureus as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • oriC polynucleotide or polypeptide are naturally occurring variants of an oriC polynucleotide or polypeptide.
  • methods for producing the aforementioned oriC polynucleotides or polypeptides are provided.
  • inhibitors to such oriC polynucleotide or polypeptides useful as antibacterial agents, including, for example, antibodies.
  • products, compositions and methods for assessing oriC activity for treating disease, for assaying genetic variation, and for administering an oriC polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Staphylococcus aureus bacteria.
  • methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of an oriC polypeptide or polynucleotide of the invention comprising: contacting an oriC polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the oriC polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the oriC polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the oriC polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the oriC polypeptide or polynucleotide.
  • oriC agonists and antagonists preferably bacteriostatic or bacteriocidal agonists and antagonists.
  • compositions comprising an oriC polynucleotide or an oriC polypeptide for administration to a cell or to a multicellular organism.
  • a computer readable medium having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of SEQ ID NO. 1 ; an oriC polypeptide; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO.
  • a set of oriC polypeptide sequences a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1 ; a data set representing a polynucleotide sequence encoding an oriC polypeptide sequence; a polynucleotide comprising the sequence of SEQ ID NO. 1 ; an oriC polypeptide; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO. 1 ; a set of oriC polypeptide sequences; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing an oriC polypeptide sequence in a computer readable medium; and comparing said oriC polypeptide sequence to at least one polynucleotide or oriC polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of SEQ ID NO. 1 in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1 in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or oriC polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of SEQ ID NO. 1 in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
  • Figure 1 shows a schematic of a proposed assay to screen for agonists and antagonists of OriC function, as more fully described elsewhere herein.
  • Figure 2 shows an embodiment of the S. aureus OriC region with motifs underlined and boxed, as more fully described elsewhere herein.
  • Figure 3 shows a schematic of the S. aureus genome structure of the stretch comprising the
  • Figure 4 shows a schematic of truncations performed on the 5. aureus OriC to elucidate its function, as more fully described elsewhere herein.
  • Figure 5 shows a graphical illustration of the results of a 5. aureus chromosomal replication assay, as more fully described elsewhere herein. DESCRIPTION OF THE INVENTION
  • the invention relates to oriC polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to oriC polypeptides and polynucleotides of a oriC polynucleotide of Staphylococcus aureus, which is related by nucleic acid sequence homology to other prokaryotic origins of replication.
  • the invention relates especially to oriC having the nucleotide sequence set out in Table 1 as SEQ ID NO: 1.
  • a deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY, Scotland on 11 September 1995 and assigned NCIMB Deposit No. 40771, and referred to as Staphylococcus aureus WCUH29 on deposit. .
  • the Staphylococcus aureus strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain.”
  • the deposited strain contains the full length oriC origin.
  • the sequence of the oriC polynucleotides contained in the deposited strain, as well as the amino acid sequence of the oriC polypeptides, are controlling in the event of any conflict with any description of sequences herein.
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure.
  • the strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 112.
  • a license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
  • an isolated nucleic acid molecule encoding a mature oriC polypeptide expressible by the Staphylococcus aureus WCUH 29 strain contained in the deposited strain. Further provided by the invention are oriC nucleotide sequences of the DNA in the deposited strain. Also provided by the invention are oriC polypeptide sequences isolated from the deposited strain and amino acid sequences derived therefrom.
  • Another aspect of the invention relates to isolated polynucleotides, including the full length oriC origin of replication and polynucleotides closely related thereto and variants thereof.
  • a polynucleotide of the invention may be obtained using standard cloning and screening methods, such as cloning from bacteria using Staphylococcus aureus WCUH 29 cells as starting material, followed by obtaining a full length clone.
  • a polynucleotide sequence of the invention such as a sequence given in Table 1 [SEQ ID NO: l]
  • a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions.
  • such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70).
  • the polynucleotide set out in Table 1 [SEQ ID NO:l] was discovered in a DNA library derived from Staphylococcus aureus WCUH 29.
  • the DNA sequence set out in Table 1 [SEQ ID NO: 1] contains protein binding motifs capable of binding oriC polypeptides.
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence in Table 1 [SEQ ID NO:l]. Also provided by the invention is an oriC polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature oriC polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence.
  • the invention also includes polynucleotides of the formula:
  • Ri and R3 is independently any nucleic acid residue
  • m is an integer between 1 and 3000 or zero
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1.
  • R 2 is oriented so that its 5' end residue is at the left, bound to R j and its 3' end residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1 may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • X and Y together define a covalent bond the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary.
  • m and/or n is an integer between 1 and 1000.
  • polynucleotides of the inventions are derived from Staphylococcus aureus, however, they may preferably be obtained from organisms of the same taxonomic genus. They may also be obtained, for example, from organisms of the same taxonomic family or order.
  • polynucleotides that are at least 70% identical over their entire length to a polynucleotide having the sequence set out in Table 1 [SEQ ID NO:l], and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide having the sequence set out in Table 1 [SEQ ID NO.l].
  • polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred.
  • those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
  • Another preferred embodiment is a minimal sequence necessary to confer replicative competence to an ErmE gene in S. aureus, for example, the sequence of SEQ ID NO:l.
  • Preferred embodiments are oriC polynucleotides that retain substantially the same biological function or activity as the oriC polynucleotide of Table 1 [SEQ LD NO:l].
  • the invention further relates to polynucleotides that hybridize to the herein above- described sequences.
  • the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions and “stringent hybridization conditions” mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C.
  • Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein.
  • the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete origin for a polynucleotide sequence set forth in SEQ ID NO: 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or a fragment thereof; and isolating said DNA sequence.
  • Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
  • polynucleotides of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding oriC and to isolate cDNA and genomic clones of other origins that have a high sequence identity to the oriC origin.
  • Such probes generally will comprise at least 15 bases.
  • such probes will have at least 30 bases and may have at least 50 bases.
  • Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
  • the coding region of the oriC origin may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO: 1] to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of an origin of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the polynucleotides and oriC polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays.
  • Polynucleotides of the invention that are oligonucleotides derived from the sequence of Table 1 [SEQ ID NO: 1] may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • the invention also provides polynucleotides that may encode an oriC polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature oriC polypeptide (when the mature form has more than one oriC polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • a precursor protein, having the mature form of the oriC polypeptide fused to one or more prosequences may be an inactive form of the oriC polypeptide.
  • prosequences When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • N may also be used in describing certain polynucleotides of the invention. “N” means that any of the four DNA or RNA bases may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a base that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.
  • a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the oriC polypeptide.
  • the invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of oriC polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • bacterial cells such as streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, staphylococci, enteroco
  • Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express an oriC polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
  • appropriate secretion signals may be incorporated into the expressed oriC polypeptide. These signals may be endogenous to the oriC polypeptide or they may be heterologous signals.
  • OriC polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the oriC polypeptide is denatured during isolation and or purification.
  • This invention is also related to the use of the oriC polynucleotides of the invention for use as diagnostic reagents. Detection of oriC in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "individual(s)"), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the oriC origin may be detected at the nucleic acid level by a variety of techniques. Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin.
  • Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis.
  • RNA, cDNA and genomic DNA may also be used in the same ways.
  • characterization of the species and strain of prokaryote present in an individual may be made by an analysis of the genotype of the prokaryote origin. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to labeled oriC polynucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or a chemical cleavage method. See, e.g., Cotton et al., Proc. Nad. Acad. ScL, USA, 85: 4397-4401 (1985).
  • RNA carrying mutations or polymorphisms (allelic variations) in the origin of the invention may also be detected at the DNA or RNA level by a variety of techniques, to allow for serotyping, for example.
  • RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
  • RNA, cDNA or genomic DNA may also be used for the same purpose, PCR or RT-PCR.
  • PCR primers complementary to a nucleic acid sequence of oriC can be used to identify and analyze mutations. Examples of representative primers are shown below in Table 2. Table 2 Primers for amplification of oriC polynucleotides SEQ ID NO PRIMER SEQUENCE
  • the invention also includes primers of the formula: X-(R 1 ) m -(R 2 )-(R 3 )n-Y wherein, at the 5' end of the molecule, X is hydrogen or a metal, and at the 3' end of the molecule, Y is hydrogen or a metal, Rj and R3 is any nucleic acid residue, m is an integer between 1 and 20 or zero , n is an integer between 1 and 20 or zero, and R 2 is a primer sequence of the invention, particularly a primer sequence selected from Table 2.
  • R 2 is oriented so that its 5' end residue is at the left, bound to R j and its 3' end residue is at the right, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer being complementary to a region of a polynucleotide of Table 1.
  • m and/or n is an integer between 1 and 10.
  • the invention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end.
  • These primers may be used for, among other things, amplifying oriC DNA isolated from a sample derived from an individual.
  • the primers may be used to amplify the origin isolated from an infected individual such that the origin may then be subject to various techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections by Staphylococcus aureus, comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1].
  • Increased or decreased expression of oriC polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • a diagnostic assay in accordance with the invention for detecting over- expression of oriC protein compared to normal control tissue samples may be used to detect the presence of an infection, for example.
  • Assay techniques that can be used to determine levels of an oriC protein, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the polynucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to, and can hybridize with, a particular location on an individual microbial chromosome, particularly a Staphylococcus aureus chromosome.
  • the mapping of relevant sequences to a chromosome according to the present invention is an important first step in correlating those sequences with origin associated with microbial pathogenicity and disease, or to chromosomal regions critical to the growth, survival and/or ecological niche. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data.
  • Such data are found in, for example, microbial genomic sequences available on the World Wide Web.
  • the relationship between origins and microbial pathogenicity, disease, or to genome regions critical to the growth, survival and/or ecological niche that have been mapped to the same chromosomal region are then identified using methods to define a genetic relationship between the origin and another origin or phenotype, such as by linkage analysis (coinheritance of physically adjacent genes).
  • RNA or genomic sequence between microbes of differing phenotypes can also be determined. If a mutation or sequence is observed in some or all of the microbes of a certain phenotype, but not in any microbes lacking that phenotype, then the mutation or sequence is likely to be the causative agent of the phenotype. In this way, chromosomal regions may be identified that confer microbial pathogenicity, growth characteristics, survival characteristics and/or ecological niche characteristics.
  • the polynucleotides and polynucleotides of the invention may be used as reagents for differential screening methods.
  • differential screening and differential display methods known in the art in which the polynucleotides and oriC polypeptides of the invention may be used.
  • the differential display technique is described by Chuang et al., 1. Bacte ⁇ ol. 775:2026-2036 (1993). This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly-primed RT-PCR. By comparing pre-infection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF 'unknowns'.
  • IVET In Vivo Expression Technology (IVET) is described by Camilli et al, Proc. Nat'l. Acad. Sci. USA. 97:2634-2638 (1994). IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role in infection. ORF identified by this technique are implied to have a significant role in infection establishment and/or maintenance. In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombinase gene in a plasmid vector. This construct is introduced into the target organism which carries an antibiotic resistance gene flanked by resolvase sites.
  • the resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance.
  • the chromosomal fragment carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally upregulated during infection. Sequencing upstream of the recombinase gene allows identification of the up regulated gene.
  • RT-PCR may also be used to analyze gene expression patterns.
  • messenger RNA is isolated from bacterial infected tissue, e.g., 48 hour murine lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs.
  • the determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacterials.
  • the bacterial mRNA preparation need not be free of mammalian RNA. This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived in the bacterium (in the order of 2 minute halflives).
  • the bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA.
  • the process is optimised by finding those conditions which give a maximum amount of Staphylococcus aureus 16S ribosomal RNA as detected by probing Northerns with a suitably labelled sequence specific oligonucleotide probe.
  • a 5' dye labelled primer is used in each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles.
  • the PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI).
  • oriC polypeptides of the invention or variants thereof, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such oriC polypeptides.
  • Antibodies as used herein includes monoclonal and polyclonal antibodies, chimeric, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunolglobulin expression library.
  • Antibodies generated against the oriC polypeptides of the invention can be obtained by administering the oriC polypeptides or epitope-bearing fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols.
  • any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
  • phage display technology may be utilized to select antibody genes with binding activities towards the oriC polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-oriC or from naive libraries (McCafferty, J. et al, ( 1990), Nature 348, 552-554; Marks, J. et al., ( 1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., ( 1991) Nature 352, 624-628). If two antigen binding domains are present each domain may be directed against a different epitope - termed ⁇ jispecific' antibodies.
  • antibodies may be employed to isolate or to identify clones expressing the oriC polypeptides to purify the oriC polypeptides by affinity chromatography.
  • antibodies against an oriC polypeptide may be employed to treat infections, particularly bacterial infections.
  • OriC polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of this invention.
  • the term "antigenically equivalent derivative” as used herein encompasses an oriC polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or oriC polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host.
  • the term “immunologically equivalent derivative” as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
  • the oriC polypeptide such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken.
  • the fusion protein may provide stability to the oriC polypeptide.
  • the antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the protein or oriC polypeptide, or an antigenically or immunologically equivalent oriC polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
  • the antibody or variant thereof is modified to make it less immunogenic in the individual.
  • the antibody may most preferably be "humanized”; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al. (1986), Nature 321 , 522-525 or Tempest et al., (1991) Biotechnology 9, 266-273.
  • a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1 :363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed with specific protein carriers (Wu et al., I Biol Chem.
  • OriC polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).
  • the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of oriC polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and/or bacteriocidal.
  • the method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising oriC polypeptide and a labeled substrate or ligand of such oriC polypeptide is incubated in the absence or the presence of a candidate molecule that may be an oriC agonist or antagonist.
  • the ability of the candidate molecule to agonize or antagonize the oriC polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.
  • Molecules that bind gratuitously, i.e., without inducing the effects of oriC polypeptide are most likely to be good antagonists.
  • Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system.
  • Reporter systems that may be useful in this regard include but are not limited to colorimetric labeled substrate converted into product, a reporter origin that is responsive to changes in oriC polynucleotide or oriC polypeptide activity, and binding assays known in the art.
  • oriC antagonists are a competitive assay that combines oriC and a potential antagonist with oriC-binding molecules, recombinant oriC binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
  • oriC can be labeled, such as by radioactivity or a colorimetric compound, such that the number of oriC molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
  • Potential antagonists include small organic molecules, peptides, oriC polypeptides and antibodies that bind to a polynucleotide or oriC polypeptide of the invention and thereby inhibit or extinguish its activity.
  • Potential antagonists also may be small organic molecules, a peptide, an oriC polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing oriC-induced activities, thereby preventing the action of oriC by excluding oriC from binding.
  • Potential antagonists include a small molecule that binds to and occupies the binding site of the oriC polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.
  • small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.
  • Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules).
  • Preferred potential antagonists include compounds related to and variants of oriC.
  • Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds.
  • an oriC protein upon expression, can be used as a target for the screening of antibacterial drugs.
  • the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
  • the invention also provides the use of the oriC polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection.
  • the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matrix proteins on indwelling devices or to extracellular matrix proteins in wounds; to block oriC protein- mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al., Infect. Immun.
  • the antagonists and agonists of the invention may be employed, for instance, to inhibit and treat diseases.
  • H. pylori Helicobacter pylori bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France; http://www.uicc.ch/ecp/ecp2904.htm).
  • the international Agency for Research on Cancer recently recognized a cause-and-effect relationship between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen.
  • Preferred antimicrobial compounds of the invention should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. p>y/o ⁇ ' -induced cancers, such as gastrointestinal carcinoma. Such treatment should also cure gastric ulcers and gastritis.
  • Vaccines agonists and antagonists of oriC found using screens provided by the invention, particularly broad-spectrum antibiotics.
  • Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with oriC, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection. Also provided are methods whereby such immunological response slows bacterial replication.
  • Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of oriC, or a fragment or a variant thereof, for expressing oriC, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not.
  • an immunological response such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not.
  • One way of administering the origin is by accelerating it into the desired cells as a coating on particles or otherwise.
  • Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
  • a further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to an oriC or protein coded therefrom, wherein the composition comprises a recombinant oriC or protein coded therefrom comprising DNA which codes for and expresses an antigen of said oriC or protein coded therefrom.
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.
  • An oriC polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties.
  • fused recombinant protein preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta- galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof.
  • an antigenic co-protein such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta- galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof.
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.
  • the co-protein may be attached to either the amino or carboxy terminus of the first protein.
  • compositions particularly vaccine compositions, and methods comprising the oriC polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
  • kits using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection with Staphylococcus aureus will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Staphylococcus aureus infection, in mammals, particularly humans.
  • the oriC polynucleotide or polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria.
  • the invention also includes a vaccine formulation which comprises an immunogenic recombinant protein or polynucleotide of the invention together with a suitable carrier. Since the protein or polynucleotide may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation insotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • compositions for purposes of compositions, kits and administration
  • the invention also relates to compositions comprising the oriC polynucleotide or the oriC polypeptides discussed above or their agonists or antagonists.
  • the oriC polypeptides and/or polynucleotide of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject.
  • Such compositions comprise, for instance, a media additive or a therapeutically effective amount of an oriC polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • OriC polypeptides and/or polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • the pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
  • the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams.
  • Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
  • Such carriers may constitute from about 1 % to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
  • the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg.
  • the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • a vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.
  • a suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
  • polynucleotide and oriC polypeptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algorithms.
  • polynucleotide of the invention and “polynucleotide sequence of the invention” mean any detectable chemical or physical characteristic of a polynucleotide of the invention that is or may be reduced to or stored in a computer readable form. For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data.
  • oriC polypeptide of the invention and "oriC polypeptide sequence of the invention” mean any detectable chemical or physical characteristic of an oriC polypeptide of the invention that is or may be reduced to or stored in a computer readable form. For example, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data.
  • the invention provides computer readable medium having stored thereon sequences of the invention.
  • a computer readable medium having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of a polynucleotide of the invention; an oriC polypeptide comprising the sequence of an oriC polypeptide sequence of the invention; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of a polynucleotide sequence of the invention; a set of oriC polypeptide sequences wherein at least one of said sequences comprises the sequence of an oriC polypeptide sequence of the invention; a data set representing a polynucleotide sequence comprising the sequence of polynucleotide sequence of the invention; a data set representing a polynucleotide sequence encoding an oriC polypeptide sequence comprising the sequence of an oriC polypeptide sequence of the invention; a polynucleotide comprising the sequence of
  • the computer readable medium can be any composition of matter used to store information or data, including, for example, commercially available floppy disks, tapes, hard drives, compact disks, and video disks.
  • methods for the analysis of character sequences particularly genetic sequences.
  • Preferred methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, and sequencing chromatogram peak analysis.
  • a computer based method for performing homology identification, said method comprising the steps of: providing an oriC polypeptide sequence comprising the sequence of an oriC polypeptide of the invention in a computer readable medium; and comparing said oriC polypeptide sequence to at least one polynucleotide or oriC polypeptide sequence to identify homology.
  • a computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing a polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or oriC polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing an oriC polypeptide sequence comprising the sequence of an oriC polypeptide of the invention in a computer readable medium; and comparing said oriC polypeptide sequence to at least one polynucleotide or oriC polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
  • Disease(s) means and disease caused by or related to infection by a bacteria, including disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric absces, toxic shock syndrome), skin (
  • Identity is a relationship between two or more oriC polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between oriC polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
  • Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., 1. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST).
  • oriC polypeptide sequence comparison includes the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 ( 1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4
  • a program useful with these parameters is publicly available as the "gap” program from Genetics Computer Group, Madison WI.
  • the aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: l, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO: l
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: l, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO: l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO: 1 , or:
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleic acids in SEQ ID NO:l
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or an oriC polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or oriC polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein.
  • a polynucleotide or oriC polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotide(s) include, without limitation, single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • the term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)” also embraces short polynucleotides often referred to as oligonucleotide(s).
  • Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
  • a given oriC polypeptide may contain many types of modifications. Modifications can occur anywhere in an polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, se
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • Variant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of an oriC polypeptide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by a polynucleotide sequence of the invention, as discussed below.
  • a typical variant of an polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • Example 1 Strain selection, Library Production and Sequencing The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: l] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coll The sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: l . Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.
  • Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods.
  • Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures. DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli infected with the packaged library. The library is amplified by standard procedures. Method 2
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures.
  • EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library.
  • the library is amplified by standard procedures.
  • the following assay is used to examine in vitro S. aureus OriC-dependent replication (see Figure 1): An extract prepared from S. aureus, for example S. aureus strain RN4220, is supplied to a plasmid carrying a copy of OriC in a reaction including exogenous radiolabeled deoxynucleotides (dATP, dTTP, dGTP and dCTP), magnesium chloride, ATP, and an ATP regenerating system. The reaction is stopped and the products precipitated with trichloroacetic acid, and then filtered. Scintillation counting of the dried filter gives the level of de novo replication. This reaction is sensitive to rifampicin; RNA polymerase is required for chromosomal initiation under these conditions. The reaction is dependent upon OriC; the parental vector pBS-ERM gives background levels of radionucleotide incorporation.
  • dATP exogenous radiolabeled deoxynucleotides
  • dTTP
  • Example 4 In Vitro S. aureus Or C-dependent DNA Replication After subcloning this OriC sequence with an erythromycin-resistance determinant and a ColEl origin, the resulting shuttle vector readily transformed both E. coli and S. aureus. Truncation analyses revealed that the structure of the S. aureus origin is similar to that of the B. subtilis origin: two clusters of DnaA boxes flank the dnaA gene, and an A/T rich sequence is upstream of the whole (see Figure 3). Both upstream and downstream DnaA boxes are preferred for a viable origin in vivo. The constructs were further tested in a reconstituted in vitro replication system using a partially purified cellular extract.
  • the reaction depends upon DNA supercoiling and a complete OriC sequence; only background replication resulted from templates containing truncations of the origin.
  • the reaction was sensitive to rifampicin, suggesting a role for RNA polymerase in the initiation of replication.
  • rifampicin suggesting a role for RNA polymerase in the initiation of replication.
  • a demonstration of a sensitivity of the assay to either novobiocin or anilinouracils has been attempted but has not shown significant sensitivity.

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  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des polypeptides et des polynucléotides oriC, ainsi que des méthodes permettant de produire ces polypeptides et polynucléotides oriC par des techniques de recombinaison. Cette invention concerne également des méthodes d'utilisation de ces polypeptides et polynucléotides oriC, de manière à détecter par criblage des composés bactéricides.
PCT/US1999/014487 1998-06-24 1999-06-24 Origine de replication et ses methodes d'utilisation WO1999067391A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2000556033A JP2002518049A (ja) 1998-06-24 1999-06-24 複製起点および使用方法
EP99933582A EP1088074A1 (fr) 1998-06-24 1999-06-24 Origine de replication et ses methodes d'utilisation

Applications Claiming Priority (4)

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US9046798P 1998-06-24 1998-06-24
US11746999P 1999-01-27 1999-01-27
US60/090,467 1999-01-27
US60/117,469 1999-01-27

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WO1999067391A1 WO1999067391A1 (fr) 1999-12-29
WO1999067391A9 true WO1999067391A9 (fr) 2000-03-16

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WO1999067391A1 (fr) 1999-12-29
JP2002518049A (ja) 2002-06-25

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